Methylation patterns of aquatic humic substances determined by13C NMR spectroscopy

Org. Geochem. Vol. I1, No. 3, pp. 123-137, 1987 Printed in Great Britain. All rights reserved

0146-6380/87 $3.00+0.00 Pergamon Journals Ltd

Methylation patterns of aquatic humic substances determined by 13C NMR spectroscopy KEVIN A. THORN1, CORNELIUSSTEELINK2 and ROBERT L. WERSHAWt tU.S. Geological Survey, Box 25046, MS 407, Federal Center, Denver, CO 80225 and 2Department of Chemistry, University of Arizona, Tucson, AZ 85721, U.S.A. (Received 22 April 1986; accepted 2 December 1986)

AIl~i'act--13CNMR spectroscopy is used to examine the hydroxyl group functionality of a series of humic and fulvic acids from different aquatic environments. Samples first are methylated with 13C-labeled diazomethane. The NMR spectra of the diazomethylated samples allow one to distinguish between methyl esters of carboxylic acids, methyl ethers of phenolic hydroxyls, and methyl ethers of phenolic hydroxyls adjacent to two substituents. Samples are then permethylated with K3C-labeledmethyl iodide/Nail. ~3C NMR spectra of permethylated samples show that a significant fraction of the hydroxyl groups is not methylated with diazomethane alone. In these spectra methyl ethers of carbohydrate and aliphatic hydroxyls overlap with methyl ethers of phenolic hydroxyls. Side reactions of the methylation procedure including carbon methylation in the CHjI/NaH procedure, are also examined. Humic and fulvic acids from bog, swamp, groundwater, and lake waters show some differences in their distribution of hydroxyl groups, mainly in the concentrations of phenolic hydroxyls, which may be attributed to their different biogeoehemicai origins. Key words: aquatic humic substances, fulvic acid, humic acid, 13C NMR spectroscopy, methylation,

diazomethane, hydroxyl group functionality

INTRODUCTION

In recent years, direct observation by 13C N M R spectroscopy of methyl esters and methyl ethers formed during the methylation of humic substances has demonstrated potential for obtaining more accurate data on the hydroxyl group functionality of humic and fulvic acids than the traditional wet chemical methods of methylation analysis. Ogner (1979), Steelink et al. (1983), Gonzalez-Vila et aL (1983), Thorn (1984) and Preston and Schnitzer (1984), have reported natural abundance ~3C N M R spectra of methylated humic and fulvic acids. Mikita et al. (1981) and Wershaw et aL (1981) reported 13C N M R spectra of humic and fulvic acids methylated with t3C-enriched reagents. All these spectra exhibited peaks representing methyl esters of carboxylic acids and methyl ethers of phenolic, aliphatic, and carbohydrate hydroxyls. The specific information on the hydroxyl group functionality derived from t3C N M R spectra of methylated samples is not readily discernible from the ~3C N M R spectra of underivatized samples. For example, in natural abundance ~3C N M R spectra of underivafized humic substances, carboxylic acid carbons can overlap with ester, amide, and laetone carbons (165-180 ppm); phenolic carbons overlap with other non-protonated aromatic carbons (135-165ppm); carbohydrate carbons may overlap with ether and other alcoholic carbons (60-75 ppm). Of the published spectra referred to above, only a few have been of aquatic samples, primarily because o.o. It/~^

123

the isolation of aquatic humic and fulvic acids in sufficient quantities for detailed chemical analyses is difficult and tedious. More accurate information on the nature of the hydroxyl groups of aquatic humic substances is needed to understand better the structure of these materials and, therefore, to understand better their geochemical and environmental properties: complexation of metals, the concentration of organic contaminants, the interaction with oxidizing agents, etc. The procedure described by Mikita et al. (1981) is applied here to a series of aquatic humic and fulvic acids, both to ascertain whether any differences could be observed in the hydroxyl group functionality in humic substances from different aquatic environments, and to examine the methylation procedure itself in more detail. Carbon-13 enriched methylating reagents are used so that smaller amounts of the difficult to isolate aquatic humic samples can be used for N M R analysis. The use of enriched reagents offers another advantage: one can more clearly monitor side reactions. Tl~e concentration of 13C in the enriched reagents is 100 times the concentration of the naturally abundant 13C in the humic substances. With the number of transients collected on the C-13 methylated samples in this study, only the carbons which are added on to the humic molecules appear in the spectra; there is no overlap between the carbons added on and the naturally abundant ~3C atoms in the humic materials. In addition to aquatic samples, spectra of a soil humic acid, a soil fulvic acid, and two commercial humic acids are presented for comparison.

124

KEwN A. THORN et aL

In the original study of Mikita et al. (1981), NMR spectra were recorded after the two-step derivatization procedure was performed. It will be shown in this paper that more structural information can be obtained by recording NMR spectra after each step of the derivatization procedure. EXPERIMENTAL

Materials Sources of aquatic humic samples. The Williams Fork Reservoir is a man-made mesotrophic reservoir, elevation 2200m, located about l l2km westnorthwest of Denver, Colo, in the Rocky Mountains (LaBaugh and Winter, 1984). The Kassler water treatment plant, located in Waterton, Colo, southwest of Denver, holds water from the Dillon Reservoir in Dillon, Colo. Thoreau's Bog, located near Concord, Mass., is an ombrotrophic floating sphagnum bog developed in a glacial kettlehole surrounded by a red maple forest (Hemond, 1980, 1982; McKnight et al., 1985). Merrill Lake is an oligotrophic lake located in the Cascade mountains 10 miles southwest of Mt St Helens in Washington (McKnight et aL, 1984). The Biscayne Aquifer is located in Dade County, Fla, and has been described by Thurman (1985a, in Aiken et aL). The Suwannee River drains the Okeefenokee Swamp near Fargo, Ga (Cohen et aL, 1984). Isolation of aquatic samples. The Williams Fork, Thoreau's Bog, and Merrill Lake fulvic acids were isolated according to the method of Leenheer (198 I). The Waterton and Biscayne fulvic and the Suwannee humic and fulvie acids were isolated according to the method of Thurman and Malcolm (1981). The Suwannee humic and fulvic acids are the International Humic Substances Society (IHSS) standard samples. Source and isolation of soil samples. The isolation of the North Carolina humic acid from the Bh horizon of a North Carolina coastal plain sandy soil has been described by Malcolm (1964). The Armadale fulvic acid, isolated from the Bh horizon of a Prince Edward Island (Canada) podzol, was purchased from Dr Cooper H. Langford, Department of Chemistry, Concordia University, Montreal, Quebec. Preparation of commercial humic samples. Aldrich* humic acid was purchased from Aldrich Chemical Co. as the sodium salt. The sodium humate was H-saturated by passing it through a Dowex MSC-1 cation exchange resin and then freeze dried. Fluka humic acid was purchased from Fluka Chemical Corp. The Fluka humic acid was dissolved in 1.0 N NaOH under N2, hydrogen saturated as with the Aldrich, and then freeze dried. After these treatments, both the Aldrich and Fluka humic acids could be solubilized completely in DMF.

13C-enriched reagents. The N-methyl-13C-Nnitroso-p-toluenesulphonamide (92.1 at.% t3C) and t3C-iodomethane (99.0 at.% 13C) were purchased from MSD Isotopes, St Louis, Mo. Methylation procedure Approximately 50 mg of the H-saturated humic or fulvic acid was dissolved in 30-50 ml of distilled and dried DMF, and methylated with ethereal diazomethane, generated from 0.5g (2.33mmol) of N-methyl-~3C-N-nitroso-p-toluenesulphonamide, 92.1 at.% 13C. After the reaction was completed, the excess diazomethane was allowed to decompose overnight, and ether and DMF were then removed from the product with vacuum distillation. The product was then redissolved in 0.5-1.0 ml of DMF-d7 for NMR analysis (5 ram NMR tube). In some cases the diazomethylated product was soluble in CDCI3. After NMR analysis, the deuterated solvent was removed with vacuum distillation, and the product redissolved in a stoppered flask with approx. 3040 ml of DMF and stirred. The flask was blanketed in N2 and charged with 0.2 g Nail (0.4 g 50% mineral oil dispersion washed in sintered glass funnel with dried hexane) and 0.2ml ~3CH3I (3.21 retool), 99 at.% ~3C. The reaction was allowed to run overnight; then excess sodium hydride was quenched with methanol/methyl iodide. The product was extracted into chloroform from water. The chloroform extract was washed with water until free of DMF. The chloroform was evaporated off, and the permethylated humic was redissolved in 0.5-1.0 ml CDCI3 for NMR analysis (5 mm NMR tube).

NMR spectroscopy Carbon-13 NMR spectra were recorded on a Varian FT80A NMR spectrometer at 20.0 MHz with the following acquisition parameters: 5000 Hz (250 ppm) spectral width; 45 ° pulse angle; 1.023 sec acquisition time; no pulse delay; continuous broadband decoupling; -1.00 sensitivity enhancement. Deuterated solvent peaks served as internal references. Chemical shifts are expressed with respect to TMS in ppm. Because the spin lattice relaxation times and NOE (Nuclear Overhauser Enhancement) factors of the methyl ester and methyl ether peaks are very similar, the acquisition parameters employed here result in quantitatively accurate spectra. This was proven by comparing spectra acquired under these parameters to spectra acquired with 6 sec pulse delays and inverse gated decoupling. In several of the samples, both after diazomethylation and after permethylation, spectra recorded under the two different sets of acquisition parameters were identical (Thorn, 1984). RESULTS AND DISCUSSION

*Any use of trade names is for descriptive purposes only and does not constitute endorsement by the U.S. Geological Survey.

Methylation with diazomethane The 13C NMR spectra of the aquatic fulvic and

Methylation patterns of aquatic humic substances determined by ~3C NMR humic acids, after methylation with diazomethane and after permethylation with CH3I/NaH, are shown in Figs 1 and 2. The soil and commercial samples are in Fig. 3. A pattern of 3 distinct peaks, at 52, 56 and 61 ppm appears for most of the samples, aquatic, soil and commercial, after methylation with diazomethane. The chemical shifts of various methylated model compounds are illustrated in Fig. 4. The peak at 52ppm represents methyl esters of carboxylic acids. Interpretation of the peaks at 56 and 61 ppm must be correlated with the reactivity of diazomethane. In the absence of a Lewis acid or other catalyst, such as BF3 or silica gel, diazomethane will methylate carboxylic acids, enolic hydroxyls, and phenolic hydroxyls, but not carbohydrate or aliphatic hydroxyls (Black, 1983). Since no catalysts were used in the diazomethylation step, the peaks at 56 and 61 ppm cannot be assigned to the methyl ethers of carbohydrate or aliphatic hydroxyls. These peaks, therefore, must be assigned to the methyl ethers of enolic and phenolic hydroxyls. Methyl ethers of enolic hydroxyls would appear in the peak at 56 ppm; methyl ethers of phenolic hydroxyls would appear in both the 56 and 61 ppm peaks. The peak at 61 ppm represents the methyl ethers of phenolic hydroxyls adjacent to two substituents, where the ring juncture of a fused aromatic ring structure can count as a substituent. Examples of such hydroxyls include the 4-hydroxyl of gallic acid, the hydroxyl of 2-methyl-l-naphthol, the 3-hydroxyl of quercetin, and the hydroxyl of l-hydroxy-2-naphthoic acid (Fig. 4). Methyl ethers of phenols of this particular substitution pattern have been reported in methylated asphaltenes (Snape et al., 1982). The peaks at 61 ppm representing the methyl ethers of phenolic hydroxyls adjacent to two substituents are significant entities in Thoreau's Bog fulvic acid, Suwannee River humic and fulvic acids, North Carolina soil humic acid, and Aldrich and Fluka humic acids. The presence of these types of phenolic hydroxyls suggests that condensed aromatic structures, tannin-like, or flavonol-like moieties may occur in these fulvic and humic acid samples. Several of the spectra of the diazomethylated samples exhibit discrete resonances downfield of 61 ppm.

These resonances are most apparent in the Williams Fork, Biscayne, and Merrill Lake fulvic acids; vertical scale expansions of these spectra are provided in Fig. 2. These peaks are at 66.8 and 64.3 ppm for the Williams Fork, 66.6, 65.7 and 64.3 ppm in the Biscayne, and 65.5 and 64.3 ppm in the Merrill Lake samples. Although the signal to noise is low for these peaks, they were found to be reproducible, when duplicates of these samples were methylated with diazomethane, and the NMR spectra were recorded. At this point we are unable to make assignments for these peaks. Integration values for the peak areas, translated into percent of total hydroxyl, for the samples which have been methylated with diazomethane only, are listed in Table 1. No differential saturation or differential NOE effects were found among the methyl ether and methyl ester peaks in the ~3C NMR spectra; therefore, the relative percentages are accurate. The pattern of methyl ethers and esters which appears after methylation with diazomethane will be referred to as the strong acidity pattern of the fulvic and humic acids. This is perhaps an overstatement of the situation, but it will help simplify the discussion. Side reactions of diazomethane

Several side reactions of diazomethane can occur with various functional groups that may exist in humic substances. A review of the reactions of diazomethane has been published recently by Black (1983). These include 1,3 dipolar addition across carbon--carbon double bonds; C, N and S methylation; methylene insertion or epoxide formation with ketones. In the presence of metals, e.g. copper powder, ethereal solutions of diazomethane can form polymethylene (Buckley et al., 1950). These reactions are illustrated in Fig. 4 with appropriate model compounds. The laC NMR chemical shifts of the product carbons that originated from the diazomethane are listed. With the exception of 1,3 dipolar addition, no evidence exists for any of these side reactions in the laC NMR spectra of the diazomethylated samples. Resonances corresponding to C - - C H , N---CHa and S--CHa methyl groups, at approx. 25-35, 23-35 and 15ppm, respectively, re-

Table 1. Integration values for ~3C NMR spectra of diazomethylated fulvic and humic acids°

Sample Wilfiam's Fork fulvic Merrill Lake fulvic Biscayne fulvic Waterton fulvic Thoreau's Bog fulvic Suwannee fulvic Suwannee humic Armadale fulvic North Carolina humic Aldrich humic Fluka humic "Percentage of total hydroxyl.

Methyl ether of phenolic OH 60 ppm 7.7 7.4 5.8 7.4 17.9 12.6 24.0 14.2 16.8 15.6 16.0

125

Methyl ether of phenolic/ enolic OH 56 ppm 3.4 6.7 2.5 6.7 12.1 7.5 13.4 7.8 14.6 17.8 17.7

Methylester of carboxylic acid 52 ppm 88.9 85.9 91.7 85.9 70.1 79.9 62.6 78.0 68.6 66.7 66.3

126

KEVlNA. THORNet

suiting from carbon, nitrogen and sulfur methylation, are not apparent in the spectra. Peaks corresponding to the methylene groups arising from methylene insertion or epoxide formation, from approx. 43--48 ppm, are not discernible either. Polymethylene formation upon methylat/on of soil humic acids with diazomethane has been reported in one instance (Farmer and Morrison, 1960). Based on the absence of resonances at 25-30 ppm in these spectra, there is clearly no polymethylene formation with these samples upon diazomethylation. The possibility of 1,3 dipolar addition of diazomethane to humic substances has been recognized for some time. With humic acids from acid soils, Schnit-

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zer (1974) reported increases in nitrogen after mcthylation with diazomthane ranging from 0.69 to 2.40%. Spiteller (1981) methylated a soil humic acid with diazomethane and then performed an oxidative degradation on the sample. Among the reported oxidative degradation products determined via GC-MS were 1-methyl-pyrazole-(3,4)-dicarboxylic acid dimethyl ester and 2-methyl-pyrazole-(3,4)odicarboxylic acid dimethyl ester. These were not found in the degradation products when the humic acid was not methylated with diazomethane prior to oxidative degradation. In the present study, elemental analysis of the Armadale fulvic acid revealed the nitrogen content to be 0.77% N; after diazomethylation, the

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Fig. 2. Vertical scale expansion of ~3C NMR spectra of diazomethylated Williams Fork, Biscayne, and Merrill Lake fulvic acids. nitrogen content increased to 1.61% N. The substrate at 133-140 ppm for the olefinic carbons and 39 ppm site for cycloaddition therefore is approx. for the N-methyl carbons. The NMR spectra of the 0.3% mequiv./g. diazomethylated humic and fulvic acids do not exCarbon-carbon double bonds in a variety of hibit any peaks characteristic of A~-pyrazolines or configurations may undergo cycloaddition with di- N-methyl-pyrazoles. (The full 250 ppm plots of these azomethane, including ~,]~-unsaturated ketones; al- spectra exhibit no peaks at 133-140ppm.) If kcnes; conjugated alkenes; unsaturated, conjugated, A2-pyrazolines were the cycloadduct with humic samdibasic acids; and para-quinones. Pyrazolines or N- plcs, the peak of interest would overlap with the methyl pyrazoles are formed from these substrates as methyl ester band at 51-52ppm. If A2-pyrazolines illustrated in Fig. 4. The mechanisms of these reac- were formed with any of these samples, therefore, it tions have been outlined by Spiteller (1981). To is impossible to observe this in these spectra because determine whether evidence for 1,3 dipolar addition of the overlap. of diazomethane to humic substances can be observed in ~3C NMR spectra of samples methylated Permethylation with CH3I/NaH with diazomethane, the NMR chemical shifts of the After permethylation with CH~I/NaH, a pattern of diazomethane cycloadducts of a series of model four peaks at approx. 60, 59, 55 and 52 ppm emerges compounds were obtained. As illustrated in Fig. 4, in the NMR spectra of the aquatic samples. Intermaleic acid and chalcone react with diazomethane to pretation of these spectra is somewhat more compliform A2-pyrazolines; acetylene dicarboxylic acid and cated. Different types of hydroxyl groups ovedap in 1,4-naphthoquinone react to form N-methyl pyra- the region between 55 and 60ppm. The peak at zoles. Firestone (1980) has reported the formation of 55 ppm now may be comprised of methyl ethers of 3-methylpyrazoline, a A~-pyrazoline, from propylene. hydroxyls attached to the anomeric carbon of carboIn the case e r a ~- or A2-pyrazoline formation, the ~3C hydrates as well as to enolic and phenolic hydroxyls. NMR chemical shifts of the carbons originating from Methyl ethers of carbohydrate, benzylic, and alidiazomethane are at 77 and 52 ppm, respectively. In phatic hydroxyls overlap with methyl ethers of phenthe case of N-methyl pyrazole formation, with which olic hydroxyls at 59 and 60 ppm. At this point it is not two isomers can be expected, the 13C NMR chemical possible to determine what percentages of the methyl shifts of the carbons arising from diazomcthane are ethers added, onto the humic and fulvic molecules in

Methylation patterns of aquatic humic substances determined by ~C NMR the CH3I/NaH step, are due to carbohydrate, aliphatic, or weakly acidic phenolic hydroxyls. The phenol-sulfuric acid test showed that the percentage of carbohydrate carbon in the Williams Fork, Thoreau's Bog, Biscayne aquifer, and Merrill Lake fulvic acids was in all cases <2.0%; the Suwannee fulvic and humic acids were <4% (McKnight et al. 1985; Thurman, 1985). In humic and fulvic acids, furthermore, it is not known whether carbohydrates exist in configurations where the hydroxyls are free to methylate or in configurations where the hydroxyls

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are substituted and cannot bc mcthylatcd, such as in hydrolyzable tannins. The prominent peak at 56 ppm, in the spectrum of the Suwannee River humic acid, is of interest. This peak does not correspond to the methyl ether of the anomeric carbon hydroxyl of carbohydrates, since no corresponding peaks of equal intensity occur in the 58--60 ppm region. The natural abundance solution state ]3C NMR spectrum of the underivatized Suwannee humic acid acquired under quantitative conditions, demonstrated that this sample is highly

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KEVINA. THORN et al.

aromatic, with an approximate carbon distribution of 72% sp2 vs 28% sp 3 hybridized carbon (Thorn and Wershaw, 1987; Thorn, 1987). Therefore, the majority of the area under the peak at 56 ppm is most likely attributable to phenolic hydroxyl. Integration values for the spectra of the permethylated samples translated into % carboxylic acid hydroxyl and % non-carboxylic acid hydroxyl are Substrate

listed in Table 2. Because the spectral region between 55 and 60 ppm represents the methyl ethers of several types of hydroxyls, there is no point in further integrating within this region. The percentage of total hydroxyl which is carboxylic acid hydroxyl varies from 35.5% for the Suwannee River humic to 60.2% for the Armadale fulvic. The percentage of hydroxyl groups listed in Tables 6 130, ppm

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(Solvent)

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Gallic acid

e a, 56.4 ppm

".~ - ~ -o. HO O

CH,O

o

b. 60.2 ppm

Oxygen - methylation

(CDCI 3)

(2)

Quercetln

b

~

OCH3

OH

CO2H

~

a

a. 51.8 ppm

~ C O , CH,

b. 63.0 ppm

1-Hydroxy2-nephtholc acid

(CDCI 3)

Oxygen - methylation

(2)

a

~

OCH=

OH CH:

a. 60.6 ppm

Oxygen - methyletlon

(CDCI 3)

(2)

2-Methyl- 1-nephthol e O O ll II C--CH=-- C -- CH=

@..

OCH:

O

a. 55.2 ppm

C~---CH~C--CH 3

(COCI 3)

]-Benzoylacetone

Fig. 4

Enol - methyletlon

(2)

131

Methylation patterns of aquatic humic~ubstances determined by s3C NMR

1 and 2 are relative. Acquiring absolute values in terms of mequiv./g for the various hydroxyl groups could be accomplished by using internal intensity standards in the ~3C NMR spectra. From the data presented here, the area of the methyl ester peaks can be related to the values of carboxylic acid content obtained from potentiometric titration.

Substrate

Such a relationship is determined for several of the samples in Table 3. From the spectra of the diazomethylated samples the phenolic hydroxyl content is calculated by setting the area of the methyl ester peaks equal to the titration values for carboxylic acid hydroxyls. Likewise, the total non-COzH hydroxyl content is also calculated by setting the area of the

6 13C, ppm (Solvent)

Reaction product with dlazomethane

/N~

CH~S,~N~N ~ H 2-Methylthiopurine

N

8 ~-c.,

a. - 2 0 - 3 0 ppm b. - 2 5 - 3 5 ppm

CH~

b. ~ 2 5 - 3 5 ppm

CH~

b

/CH•

/CH3 CH2=CH

CH~-"CH / \ a CHz / / N

a. 75.8 ppm (CDCl 3)

a

HO=C--CH = CH -- CO2H

a. 51.3, 51.9 ppm b. 52.6 ppm

I

o

(CDCl 3)

H &2 - Pyrazoline

~CH

-- C/ " - ~ al ~ C~ ~ / N

a. 52.0 ppm (DMSO-d 6)

I

1,3-dipolar addition: formation of Zl1Pyrazoline

a

1,3 dipolar addition: formation of Ll2 Pyrazoline

(2)

1,3-dipolar addition: formation of Z~2 Pyrazoline

(2)

H &2 _ Pyrazoline

Chalcone

(4)"

a

CH~O2C~CH__c/'COzcH3

Maleic acid

Nitrogen, sulfurmethylation

(5)

Zl1 - Pyrazoline

Propylene

Carbon, nitrogenmethylatlon

(3)*

a. ~15 ppm H 6-Mercaptoimidozo 4,5-c pyridazine

Reaction type (Reference)

a

CH;O2C% sCO~CH] C~C HOzC-- C E C - - CO2H

Acetylenedlcarboxylic acid

HsC" N s N I C CH~

a. 52.0 ppm b. 134.0, 140.0 ppm

a a CH~O2C'c, = C/cO2cH3 b/ x c HsC .. ,.. N - CH~ N

(DMF-d 7)

(2)

a. 133.0, 137.0 ppm b. 39.0 ppm

1,3-dipolar addition; dehydrogenation; N-methylation: formation of N-Methyl pyrazole

N-Methyl pyrazole O H al ~..~c\ b 0

> 1,4-Napthaquinone

c. 39.0 ppm

1,3 dipolar addition; dehydrogenation; N--methylation: formation of N-Methyl pyrazole

(DMF-d 7)

(2)

N-Methyl pyrazole

Fig. 4

132

~

Substrate

A. THORN et

Reaction product with dlazomethane O II /c,~a CH2 OH,

o II

al.

6 13C, I)pcn (Solvent)

Reaction type (Reference) Methylene Insertion

a. 43.8 ppm (e, 1)

NCHI.-CH~

I I CH~ CH2

fO•bH2 b. ~48 ppm

Cyclohexanone

Epoxide formation (6)'*

I I CHz /CHz "~CH= 1. Badtler, 1983 2. Thorn, 1984

3. Lister, 1979 4. Bar,n, 1981

5. Firestone, 1977 6. Black, 1983

*Only one of several reported reaction products is illustrated here. The chemical shifts have not been reported, but are estimated from the chemical shift literature. *" The chemical shift has not been reported but is estimated from the chemical shift literature.

Fig. 4. Reaction products of model compounds with diazomethane. methyl ester peaks in the spectra o f the permethylated samples equal to the titration values. If it is assumed that carboxylic acid hydroxyls have been completely derivatized with diazomethane, then the values calcuTable 2. Integration values for ]3C NMR spectra of permethylated fulvic and humic acidsa Methyl ether of non-carboxylic Methylester of acid hydroxyP carboxylicacidb Sample 60-55 ppm 52 ppm William's Fork fulvic 49.4 50.6 Merrill Lake fulvi¢ 40.8 59.2 Biscayne fulvic 47.1 52.9 Waterton fulvic 45.8 54.2 Thorcau's Bog fulvic 45.8 54.2 Suwannee fulvic 53.5 46.5 Suwannee humic 64.7 35.3 Armadale fulvic 39.9 60.2 North Carolina humic 44.3 55.7 q~.ntage of total hydroxyl. ~Values are within ± 7%, due to different percent enrichment of methylating agents.

lated for phenolic hydroxyl represent the minimum amount of hydroxyls present in the samples. It is possible that some o f the carboxylic acids in the humic and fulvic acid samples do not methylate with diazomethane, perhaps due to strong hydrogen bonding to other groups, but are methylated in the C H s I / N a H step. If this is the case, then the area under the methyl ester peak does not represent a constant value in going from the diazomethylated sample to the permethylated sample. Table 3 indicates that the phenolic hydroxyl content of these samples ranges from 0.6 to 2.9 mequiv./g. The total non-carboxylic acid hydroxyl content ranges from 3.0 to 9.0 meqniv./g. Methylation with diazomethane can be considered a technique for determining the strongly acidic phenolic hydroxyl groups. Definite differences exist in the content of these strongly acidic hydroxyl groups among the aquatic samples judging from the spectra

Table 3. Carboxylic a~d content of aquatic humic samples determined by potentiometric titration and phenolic end total hydroxyl contents determined by NMR Total non CO2H Phenolic OH hydroxyl (mequlv./g (mequlv./g) ---CO2H, from spectra of fromspectra of (mequiv./g) diazomethylated permethylated Sample from titration samples' samplesf Merrill Lake fulvic~ 4.3 0.7 3.0 Biscayue fulvicb 6.3 0.6 5.6 Thoreau's Bo8 fulvi¢c 4.9 2.1 4.1 Suwannee fulvicd 6. l 1.5 7.0 Suwanuee humi~ 4.9 2.9 9.0 "MeKnisht et al. (1984). ~Thurman and Malcolm (1983). ~McKnisht e: al. (1985). "~Malcolm (1986). 'The values for phenolic hydroxylcontent were calculated by setting the area of the methyl ester peak equal to the titration value. This calculation is based upon the gross auumption that carboxylic acids are completelymethylated with diazomethane. IThe values for total non carboxylicacid hydroxyl were calculated by setting the area of the methyl ester peak equal to the titration value. See text.

Methylation patterns of aquatic humic substances determined by '3C NMR

133

dence for carbon alkylation in soil humic acids methylated by refluxing the humic acids in acetone with dimethyl suifate/K2CO3, based upon the appearance of C--CH3 bands in IR spectra and increases in % carbon over and above what could be attributed to oxygen methylation. Structures that can carbonmethylate with CH3I/NaH in DMF include, among others, ,,,p unsaturated ketones,/~-diketo groups, and phenols (Kittila, 1967; House, 1972). The 13C NMR chemical shifts of the C--CH3 peaks of the Cmethylated derivatives of benzoylacetone, methyl acetoacetate, dimethylmalonate, methyl phenylacetate, and ortho-cresol are 23.1, 21.I, 21.1, 26.0 and 23.2 ppm, respectively (Fig. 5). Chemical shifts of these model compounds correlate with the observed peaks in the spectra of the permethylated humic samples. Of the possible sites for carbon methylation in these humic and fulvic acids, //-diketones and ct,/~-unsaturated ketones are less likely. The first step of the derivatization procedure, diazomethylation would tend to inactivate these structures toward carbon alkylation, fl-Diketones would be converted to enol-methyl ethers, and ct,//-unsaturated ketones would undergo 1,3 dipolar addition with diazomethane. There are a wide variety of potential sites for carbon alkylation, and the broadness of the C---CH3 peaks in the '3C NMR spectra suggests that in humic substances there are indeed a variety of such Carbon alkylation sites. Carbons which can carbon alkylate are In the spectra of the permethylated samples, there significant moieties in aquatic fulvic acids. The imare broad peaks of significant intensity centered at portant implication of this data is that a large fracapprox. 24 ppm. The Williams Fork fulvic acid ex- tion of the naturally occurring aliphatic methylene hibits the largest such peak. These peaks are inter- and methine carbons in the fulvic and humic acid preted as the C - - C H 3 groups resulting from carbon molecules are in activated positions, i.e. alpha to methylation of activated methylene or methine car- carbonyl carbons and aromatic rings. The phenombons. This interpretation is based on the chemical enon of carbon alkylation in humic substances is shift range of the peaks, the known reactivity of the worthy of further investigation. It is providing useful CH3I/NaH methylating system, and on the findings information on the nature of the aliphatic structures of other researchers. Schnitzer (1974) provided evi- in these materials.

of those samples after diazomethylation. The Thoreau's Bog fulvic and Suwannee River humic and fulvic acids have higher proportions of phenolic hydroxyls, especially with respect to the peaks at 61 ppm, than the Williams Fork, Waterton, Merrill Lake, and Biscayne samples. It is interesting that Thoreau's Bog and the Suwannee River (Okeefenokee Swamp) have very high DOCs and can be considered black waters. Both Thoreau's Bog and the Suwannee River have average DOCs of aprox. 35 mg C/I. The other aquatic systems have DOC levels less than 5 mg C/l, which are closer to the average DOC for most lakes, streams and groundwaters (Thurman, 1985). Also of interest is the fact that the soil humic and fulvic acids and the two commercial humic acids show patterns of strong acidity similar to the black water aquatic samples. The spectra of the Williams Fork and Waterton fulvic acids differ from the Suwannce River fulvic after diazomethylation but are almost identical after permethylation. This indicates that the Williams Fork and Waterton samples have lower amounts of strongly acidic hydroxyl groups and greater amounts of weakly acidic hydroxyl groups; whereas, the Suwannee River has relatively greater amounts of strongly acidic hydroxyl groups and lower amounts of weakly acidic hydroxyl groups.

O

O

II II R--C ~CH2--C~R 0

II RO ~ C ~

R--

i

CHaI/NaH

0

IJ CH2~C ~

CH2~CH~

CH ~

ill3

i

O

R~C~CH~C~R

+

OR

0 J ~

CHsi/Nail .~ DMF

OH

0 CH3

CH3

O

J

II

R~CH~CH~CH--C--

CH31/ NaH

o,--'r U.j

CH, O

II I IJ R~C~C~C~R I 0t3 CHs 0

I Jl I Cit3

+ R~CH~CH--C~C--

Kev]N A. T~ov.~ et al.

134

$ubstrate

Reaction product" with NaHICH31 in DMF

CH=CHICH=CH20 H

a CH~CHzCH~CHzO CH=

n-Butanol

~

--C HtO CSH~

CH2OH

Benzyl alcohol HOCH2

CH:O~I~OCH= OCH)

OH

O

C-CHt-C-CH s

a. 58.5 ppm

Oxygen- methylation

(CDCI 3)

(1)

a, 57.8 ppm

Oxygen- methylation

(CDCI 3)

(2)

a, 55,2 ppm b.-e. 59.0, 59.2, 60.4, 60.8, ppm

Oxygen - methylation

/~,

II i 'I) a

O

0

H

CHj-- C --CH= - CO2CH~

II

Dimethyl maionate

a ?H 3 --

a. 21.1 ppm CO~CH)

a/H3 a CH+ I CHiC,C- C -CO zCH~ I a CH~ a CH3

CH2-- CO zCH3

~ -

a. 23.1 ppm (CDC) 3)

CH~

CH3--C--C

Methyl acetoacetate CHzO~C-CHz -- CO~CH=

a CH 0

0

(1)

(CDCI 3)

b-e

Benzoylacetone

~ -

Reaction type (Reference)

CH3OCH2


6 ~30. I~m (8olvent)

(CDCI 3) a. 21.1 ppm

Carbon - methylation (1)

Carbon - methylatlon (1)

Carbon - methylation

(CDCI 3)

(1)

a, 26,0 ppm

Carbon - methylatlon

(CDCI 3)

(1)

a. 23.2 ppm

Carbon - methylation

(DMF-d 7)

(1)

~ -- CO=CH= a CH:

Methyl phenylacetate a

o-Cresol

1. Thorn, 1954

2. Sadtler, 1953

"Only major reaction products Illustrated

Fig. 5. Reaction products of model compounds methylated with methyl iodide/sodium hydride. Biogeochemistry

Although biogeochemical interpretations are best made from the whole complement of NMR data, including ~H NMR spectra, quantitative natural abundance :3C NMR spectra, and multiplicity sorting experiments such as APT and DEPT (Attached Proton Test, Patt and Shoolery, 1982; Distortionless Enhancement by Polarization Transfer, Doddrell et aL, 1982), some preliminary speculations can be made from the methylation data alone. In rivers, bogs and swamps, allochthonous derived humic substances are considered to predominate over autochthonous derived humic substances; in clear lakes the situation may be reversed (Steinberg and Muenster, 1985). Of the aquatic samples examined in this study, the Merrill Lake, Williams Fork and

Waterton fulvic acids would be expected to reflect greater proportions of autochthonous to allochthonous inputs than the Thoreau's Bog and Suwannee River samples. Algal derived autochthonons inputs of DOC may have a significant effect on the nature of the dissolved humic substances in Merrill Lake, Williams Fork reservoir, and the Dillon reservoir (Waterton sample) in addition to the DOC inputs from the surrounding watersheds of these lakes. In fact, the Williams Fork fulvic acid was isolated 1-2 weeks after the die off of a blue green algal bloom. The vegetation contributing to the dissolved aquatic humic substances of Thoreau's Bog is dominated by Sphagnum mosses and low growing shrubs typical of northern bogs (McKnight et al., 1985). A large variety of vegetation contributes to the dissolved humic substances of the Okeefenokee

Methylation patterns of aquatic humic substances determined by 13C NMR Swamp, including pine, maple, and cypress trees, sphagnum mosses, deciduous and evergreen shrubs, and grasses (Cohen et al., 1984). The methyl ether peaks centered at 61 ppm in the ~3C NMR spectra of the diazomethylated samples, representing phenolic hydroxyls adjacent to two substituents, may be serving as an important signature. These peaks are most prominent in the soil samples and in the Suwannee River and Thoreau's Bog samples. Several classes of naturally occurring compounds may contain these substitution patterns of phenolic hydroxyls: flavonols, tannins, both hydrolysed and condensed, and lignins. These natural products all occur in higher plants but not in lower plants such as algae and bacteria. Structural units of lignins exhibiting this substitution pattern of phenolic hydroxyl would include those units derived from sinapyl alcohol (Adler, 1977). Thus the presence of these phenolic hydroxyls in significant concentrations in the black water humic substances, the Suwannee River and Thoreau's Bog samples, may be an indicator of their predominantly allochthonous orion.

i

H2OH

CH

II

CIt

OH

Alternatively, the dissolved humic substances in Thoreau's Bog and the Okecfenokee Swamp, because of the physical settings of these aquatic environments and the very dark color of the waters, experience less exposure to sunlight compared to the Merrill Lake, Williams Fork, and Waterton fulvic acids. The Thoreau's Bog and Suwarmee River fulvic and humic acids might therefore undergo less photochemical alteration of phenolic hydroxyl compared to humic materials in other aquatic environments where there is greater exposure to sunlight. In contradiction to these two possible explanations for the greater concentrations of phenolic hydroxyls represented by the peak at 61 ppm in the black water samples (Fig. 1--A spectral), autochthonous mechanisms for the formation of these phenolic hydroxyls should not be ruled out. Wilson et al. (1983) suggested that uronlc acids may be a source of aromatic precursors to both marine and terrestrial humic

A¢

COOH

OH

materials. Popoff and Theander (1976) have shown that phenolic compounds can be formed from uronic acids at pH7 (scheme below). Each of these compounds contains a phenolic hydroxyl adjacent to two substituents which would contribute a 13C NMR peak around 61 ppm upon methylation. Thus, uronic acids derived from phytoplankton in the aquatic environment may contribute phenolic moieties which can become incorporated into fulvic and humic acids via an authochthonous pathway. With respect to the permethylation data (Figs 1 and 3--B spectra), one major difference between the soil and aquatic samples is evident: the aquatic materials are more hydroxylated than the soil samples. The Armadale soil fulvic acid and the North Carolina soil humic acid undergo the least amount of additional methylation with the NaH/CH3I step. As discussed above, the combination of diazomethylation and permethylation data suggested differences in patterns of weak and strong acidity in the Williams Fork and Waterton fulvic acids on the one hand and the Suwannee River fulvic acid on the other. Another factor, not mentioned thus far, which may effect differences in the hydroxyl group functionality of aquatic humic substances from different environments, is the nature of the surrounding soils. For example, the dissolved humic substances in the Florida Everglades, which recharges the Biscayne aquifer (Thurman, 1985a, in Aiken et al.), would be expected to have chemical characteristics similar to the humic substances of the Okeefenokee Swamp. The low content of strongly acidic phenolic hydroxyl of the Biscayne fulvic acid compared to the Suwannee River fulvic acid (Fig. I--A spectra), might possibly be explained by adsorption of a phenolic rich fraction of the humic substances in the Biscayne aquifer by clays present in the aquifer. SUMMARY

Earfier studies (sehnitzer, 1974; Stevenson, 1982) have amply demonstrated that no one methylation procedure is completely satisfactory for humic substances. Most methods either do not fully derivatize hydroxyl groups or entail significant side reactions. However, side reactions of a derivatization procedure can be viewed more in a positive than negative light, for they can provide important complementary information. Such is the case with carbon alkylation in the permethylation procedure. Methylation with diazomethane is the simplest and most straightforward method for looking at the

OH

OH

135

CH~

OH OH

136

~

A. TtlogN et al.

proportion of strongly acidic phenolic hydroxyls. To determine the ratio of carboxylic acid hydroxyl to total hydroxyl content, it is necessary to use a procedure which defivatizes all hydroxyl groups, such as the permethylation procedure used here. Shortcomings of the permethylation procedure notwithstanding, it has provided useful information on the nature of the hydroxyl groups present in aquatic humic substances. Phenolic hydroxyls are definite constituents in all of the fulvic and humic acids examined here. A significant fraction of the hydroxyl groups of aquatic humic substances are not derivatized with diazomethane. Carbons which can undergo carbon alkylation are significant moieties in aquatic humic substances. Finally, the permethylation procedure can show differences in the hydroxyl group functionality of humic and fulvic acid samples from different environments. Further research into derivatizations of functional groups of humic substances for subsequent N M R analyses should prove very fruitful. There is potential for developing new procedures which can more clearly differentiate among weakly acidic phenolic groups, and benzylic, carbohydrate, and aliphatic hydroxyls. More detailed information on the hydroxyl group functionality of humic substances not only will enable us to better understand the environmental properties of the materials, but may also provide insight into their biogeochemical origins. Acknowledgements--The authors thank D. M. McKnight, E. M. Thurman, and R. L. Malcolm of the U.S. Geological Survey for the aquatic humic samples which they provided. The helpful comments of G. R. Aiken and D. M. McKnight are also acknowledged.

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137

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